ABSTRACT

The aim of this study was to examine the inhibitory effect of the non-aromatizable androgens on FSH-stimulated aromatase activity in porcine granulosa cells. The cells were isolated from medium-sized follicles (4–6 mm) of prepubertal pigs, and cultured under chemically defined conditions in the presence of FSH (1 μg/ml, NIADDK-oFSH-S13) with and without the androgens for an initial 48-h induction period. Subsequently, the spent medium was replaced with fresh medium containing only testosterone as substrate and the cells were reincubated for a further 6 h. The conversion of this steroid to oestradiol-17β during this latter 'test' period was taken as a measure of the aromatase activity. The addition of 5α-dihydrotestosterone (DHT) into cultures of FSH-stimulated cells during the induction period resulted in a definite dose-dependent inhibition (30–70%) of the aromatase activity expressed in the test period. This inhibitory action, of the mixed non-competitive type, is characterized by a decrease in the apparent V _max and an increase in the K _m value, suggestive of an androgen inhibition of FSH-stimulated aromatase synthesis. This inhibition was also shown by the other 5α- and 5β-reduced androgens: 5β-androstanedione was the most effective, while DHT was the least. Other steroids such as pregnenolone and progesterone were inhibitory, but testosterone and diethylstilboestrol were stimulatory. These results suggest an important mechanism for the intrafollicular control of oestrogen synthesis, involving a possible reciprocal relationship between aromatase and 5α-reductase activities.

J. Endocr. (1986) 108, 335–341

Abstract

Previous studies have demonstrated the existence of several key components of the renin–angiotensin system in the pancreas. In the present study, the localization of angiotensin II receptor subtypes, type I (AT₁) and type II (AT₂), in the mouse and the rat pancreas was studied by immunocytochemistry using specific antipeptide antibodies against the second extracellular loops of AT₁ and AT₂ receptors in conjunction with confocal laser scanning microscopy. In the mouse, immunoreactivity for AT₁ and AT₂ was observed predominantly in the endothelia of the blood vessels and the epithelia of the pancreatic ductal system. Similar distribution of immunoreactivity for AT₁ and AT₂ was also observed. However, the intensity of immunoreactivity for AT₁ and AT₂ was stronger in the rat than that found in the mouse pancreas. Much weaker immunostaining for both AT₁ and AT₂, as compared with that found in ductal regions, was also found in the acini of the rodent pancreas. Together with the previous findings, the present results suggest that AT₁ and/or AT₂ receptors may play a role in regulating pancreatic functions in the rodent.

Journal of Endocrinology (1997) 153, 269–274

Abstract

Sequences from cDNA molecules encoding α₂-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human α₂C2-, α₂C4- and α₂C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either ³²P or digoxigenin and used to study the expression of α₂-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic β-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both α₂C2 and α₂C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding α₂C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each α₂-adrenoceptor subtype in sections of human pancreas. All three subtypes of α₂-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffinembedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three α₂-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple α₂-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of α₂-adrenoceptor subtype mRNA species in pancreatic β-cells was confirmed by Northern blotting of RNA extracted from the clonal β-cell line, HIT-T15. Transcripts encoding each of the three cloned α₂-adrenoceptor subtypes were detected in HIT-T15 cells.

Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with β₂-adrenoceptor mRNA revealed expression of this species in islet β-cells but not in the exocrine tissue of the pancreas.

Journal of Endocrinology (1996) 148, 531–543

Abstract

The localization and distribution of angiotensin II (Ang II) in the rat epididymis was studied using immunohistochemical and RIA techniques. The immunohistochemical results showed that Ang II-like immunoreactivity progressively increased along the length of the rat epididymis (cauda>corpus>>caput) and was predominately localized in the basal region of the epididymal epithelium. Occasionally, immunostaining of lighter intensity was also found in the apical region. The concentration of Ang II in cultured rat cauda epididymal epithelial cells was further measured by RIA. In addition to that found in cultured epithelial cells, Ang II activity was also detected in the culture medium, suggesting a secretory role of the epithelium. These findings suggest that Ang II could be derived locally from epididymal epithelium and that it could play a role in local regulation of epithelial transport and, possibly, in the maintenance of sperm function as well, by exerting its paracrine and/or autocrine effect in various regions of the epididymis.

Journal of Endocrinology (1996) 149, 217–222

SUMMARY

The administration of human menopausal gonadotrophin (HMG) followed by human chorionic gonadotrophin (HCG) stimulated the development of various numbers of follicles in patients treated for infertility. Graafian follicles from these patients were aspirated 32–33 h after the injection of HCG and the levels of steroids in the follicular fluid and matching serum samples were measured by radioimmunoassay. The follicles could not be grouped into two distinct clusters as found in patients given HCG during the menstrual cycle but a broad classification of follicles into four groups was indicated from the dendrogram. Two of the groups were similar to the ovulatory and non-ovulatory groups found previously, whereas the other two groups of follicles were more intermediate in nature. The use of a discriminant analysis showed that these two groups had clearly been stimulated by the HMG and HCG, although they were not yet fully ovulatory.

Our data indicate that the number of developing follicles is considerably increased by treatment with HMG and HCG but there is asynchrony in follicular development because the pattern of steroid synthesis differs in many follicles. The effects of this asynchronous development on oocyte maturation and disorders of the luteal phase are discussed.

SUMMARY

Human chorionic gonadotrophin (HCG) was given to patients at mid-cycle before the endogenous LH surge. Graafian follicles were aspirated 32–33 h later, before ovulation was expected, and the levels of several steroids in follicular fluid and in matching serum samples were measured by radioimmunoassay. Two types of Graafian follicle were identified at laparoscopy, based on the nature of the oocyte, granulosa cells and follicular fluid withdrawn from the follicles. Some were large, preovulatory and presumably becoming luteinized while others were generally smaller, non-ovulatory and still growing.

The concentrations of dehydroepiandrosterone (DHEA) and 17α-hydroxypregnenolone (Δ⁵ intermediates), androstenedione and testosterone were higher in non-ovulatory follicles, whereas large follicles usually contained high levels of progesterone, 17α-hydroxyprogesterone, pregnenolone and oestradiol-17β. A cluster analysis of these data grouped follicles into two distinct clusters, which accorded with their identification as ovulatory or non-ovulatory at laparoscopy.

Levels of progesterone, 17α-hydroxyprogesterone and oestradiol-17β in follicular fluid were high in preovulatory follicles in comparison with plasma. Results in two patients indicated that plasma levels of these steroids were determined by the preovulatory follicle. Levels of plasma Δ⁵ steroids were closer to follicular fluid concentrations, whereas DHEA was higher in plasma.

The role of the theca and granulosa is discussed in relation to the synthesis of progesterone and oestradiol-17β in follicles as ovulation approaches.

The steroid hormone, estrogen, plays an important role in various physiological events which are mediated via its nuclear estrogen receptors, ERalpha and ERbeta. However, the molecular mechanisms that are regulated by estrogen in the uterus remain largely unknown. To identify genes that are regulated by estrogen, the ovariectomized mouse uterus was exposed to 17beta-estradiol (E2) for 6 h and 12 h, and the data were analyzed by cDNA microarray. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include small proline-rich protein 2A, receptor-activity-modifying protein 3, inhibitor of DNA binding-1, eukaryotic translation initiation factor 2, cystatin B, decorin, secreted frizzled-related protein 2, integral membrane protein 2B and chemokine ligand 12. The expression patterns of several selected genes identified by the microarray analysis were confirmed by RT-PCR. In addition, laser capture microdissection (LCM) was conducted to determine the expression of selected genes in specific uterine cell types. Analysis of early and late responsive genes using LCM and cDNA microarray not only suggests direct and indirect effects of E2 on uterine physiological events, but also demonstrates differential regulation of E2 in specific uterine cell types. These results provide a basic background on global gene alterations or genetic pathways in the uterus during the estrous cycle and the implantation period.

Thyroid hormones (THs) are essential for normal fetal development, with even mild perturbation in maternal thyroid status in early pregnancy being associated with neurodevelopmental delay in children. Transplacental transfer of maternal THs is critical, with increasing evidence suggesting a role for 3,3′,5-tri-iodothyronine (T3) in development and function of the placenta itself, as well as in development of the central nervous and other organ systems. Intrauterine growth restriction (IUGR) is associated with fetal hypothyroxinaemia, a factor that may contribute to neurodevelopmental delay. The recent description of monocarboxylate transporter 8 (MCT8) as a powerful and specific TH membrane transporter, and the association of MCT8 mutations with profound neurodevelopmental delay, led us to explore MCT8 expression in placenta. We describe the expression of MCT8 in normal human placenta throughout gestation, and in normal third-trimester placenta compared with that associated with IUGR using quantitative reverse transcriptase PCR. MCT8 mRNA was detected in placenta from early first trimester, with a significant increase with advancing gestation (P=0.007). In the early third trimester, MCT8 mRNA was increased in IUGR placenta compared with normal samples matched for gestational age (P<0.05), but there was no difference between IUGR and normal placenta in the late third trimester. Western immunoblotting findings in IUGR and normal placentae were in accord with mRNA data. MCT8 immunostaining was demonstrated in villous cytotrophoblast and syncytiotrophoblast as well as extravillous trophoblast cells from the first trimester onwards with increasingly widespread immunoreactivity seen with advancing gestation. In conclusion, expression of MCT8 in placenta from early gestation is compatible with an important role in TH transport during fetal development and a specific role in placental development. Altered expression in placenta associated with IUGR may reflect a compensatory mechanism attempting to increase T3 uptake by trophoblast cells.

Melanocortin receptor accessory protein 2 (MRAP2) is a transmembrane accessory protein predominantly expressed in the brain. Both global and brain-specific deletion of Mrap2 in mice results in severe obesity. Loss-of-function MRAP2 mutations have also been associated with obesity in humans. Although MRAP2 has been shown to interact with MC4R, a G protein-coupled receptor with an established role in energy homeostasis, appetite regulation and lipid metabolism, the mechanisms through which loss of MRAP2 causes obesity remains uncertain. In this study, we used two independently derived lines of Mrap2 deficient mice (Mrap2 ^tm1a/tm1a ) to further study the role of Mrap2 in the regulation of energy balance and peripheral lipid metabolism. Mrap2 ^tm1a/tm1a mice have a significant increase in body weight, with increased fat and lean mass, but without detectable changes in food intake or energy expenditure. Transcriptomic analysis showed significantly decreased expression of Sim1, Trh, Oxt and Crh within the hypothalamic paraventricular nucleus of Mrap2 ^tm1a/tm1a mice. Circulating levels of both high-density lipoprotein and low-density lipoprotein were significantly increased in Mrap2 deficient mice. Taken together, these data corroborate the role of MRAP2 in metabolic regulation and indicate that, at least in part, this may be due to defective central melanocortin signalling.